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| United States Patent |
5,262,405
|
|
Girod-Vaquez
, et al.
|
November 16, 1993
|
Method of improving bronchial mucus transport
Abstract
A composition for use in the treatment of the obstruction of air ways
comprising one or more phospholipids, alone or in association with
excipients. This composition can be used in a method of treating the
obstruction of air ways in a patient.
| Inventors:
|
Girod-Vaquez; Sophie (Reims Cedex, FR);
Puchelle; Edith (Reims Cedex, FR);
Galabert; Claude (Giens - Hyeres, FR);
Zam; Jean-Marie (Reims Cedex, FR);
Pierrot; Denis (Reims Cedex, FR)
|
| Assignee:
|
Synthelabo (Le Plessis Robinson, FR);
Inserm (Paris, FR)
|
| Appl. No.:
|
658330 |
| Filed:
|
February 20, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
514/75; 514/76; 514/77; 514/78 |
| Intern'l Class: |
A61K 031/66 |
| Field of Search: |
514/75,76,77,78
|
References Cited
U.S. Patent Documents
| 4603124 | Jul., 1986 | Takei et al. | 514/78.
|
| 4765987 | Aug., 1988 | Bonte et al. | 514/78.
|
| 5032585 | Jul., 1991 | Lichterberger | 514/78.
|
Other References
von Seefeld et al., Fortschr Med 102(39) 977-981 (Oct. 18, 1984).
Schlimmer et al. Eur J. Resp. Dis Suppl., 128 (Pt. 1): 318-321 (1985).
|
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Witz; Jean C.
Attorney, Agent or Firm: Jacobson, Price, Holman & Stern
Claims
We claim:
1. A method of improving bronchial mucus transport in a patient in need
thereof which comprises administering to the patient a therapeutically
effective amount of phosphatidylglycerol (diC.sub.18:0).
2. The method according to claim 1, wherein the phosphatidylglycerol
(diC.sub.18:0) is provided in the form of a micellar suspension.
3. The method according to claim 1, wherein the phosphatidylglycerol
(diC.sub.18:0) composition is provided in the form of a liposome
suspension.
Description
The present invention relates to a pharmaceutical composition based on
phospholipids.
The integrity of the respiratory mucosa epithelium depends on a balance
between the factors of aggression and the mechanisms of defence at its
disposal. The first line of defence of the tracheo-bronchial tree is the
mucus which forms, at the surface of the respiratory epithelial cells, a
protective film which is permanently mobilised by ciliary activity.
During inflammatory bronchitis, acute or chronic, the hypersecretion of
mucus is accompanied by an alteration in its rheological properties, and
an increase in the viscosity of the mucus and in its ability to adhere to
the epithelial cells of the tracheo-bronchial wall. These abnormalities
are manifested in a decrease in the mucociliary clearance and a stasis of
mucus with bronchial obstruction. The transport of mucus by cough would
then make up for the deficiency of the muco-ciliary clearance. It is
therefore very important to preserve the efficiency of coughing, which
depends on the viscous properties and the visco-elastic properties of the
mucus and may also be greatly influenced by the interfacial properties
between the mucus and the respiratory mucosa.
Up until now, therapies to correct the hypersecretion of mucus and its
abnormalities have all developed on the basis of the fundamental concept
that the treatment of hypersecretion ought primarily to address the
alteration in the viscosity of the mucus considered as the only physical
factor responsible for the stasis of the mucus and the bronchial
obstruction. This concept has led to the development of numerous molecules
with mucolytic activity which are active in vitro or in vivo and which are
intended to normalise the hyperviscosity of the mucus.
In fact, during acute and/or chronic bronchial infections, secretions
adhere firmly to the mucosa and their elimination by the ciliary activity,
and even more by cough, can only be improved with difficulty by the
prescription of a mucolytic therapy. Mucolytic agents, whether active in
vivo or in vitro, act only on the fibrillar structure of the gel phase of
the mucus and are inactive on the surface-active properties of the mucus.
Mucus possesses natural biochemical constituents among which are included
the phospholipids which are capable of altering the mucus-mucosa
interfacial properties.
It has been shown in numerous studies, in particular in acute respiratory
distress syndrome of the new-born, that an immature lung which lacks
surfactant, that is to say surface-active substance, can be made
functional by the mere injection of artificial or natural surfactant. A
large number of studies have also shown the presence of phospholipids,
phosphatidylcholine in particular, in respiratory mucus and in
expectorations, in healthy individuals as well as in individuals suffering
from respiratory diseases accompanied by hypersecretion.
Phospholipids, including phospholipids with surface-active properties, have
been credited with the role of stabilising the small air passages. They
have also been credited, in their capacity as surface-active agents which
are present at the sol phase-gel phase interface of the mucus and by
virtue of their hydrophobic properties, with the role of regulating the
water content of the sol phase. On the other hand, despite the hypotheses
put forward, no study has shown how, by their presence in the sol
phase-gel phase interface of the mucus, these phospholipids could
effectively play a protective role towards respiratory mucosa and a
lubricating role in the transport of the gel phase on the sol phase by the
ciliary layer or by the mechanism of cough.
In bronchial pathology such as cysticfibrosis, previous studies have shown
that in expectorations, some lipid fractions can be increased and
significantly correlated with viscosity (cholesterol, glycosphingolipids,
sphingomyelin) and could therefore be considered as "rigidifying" factors.
On the other hand, it has been shown that some phospholipid fractions,
like phosphatidylglycerol, are negatively correlated with viscosity. It
appears that phosphatidylglycerol, although found in low quantities in
bronchial secretions (10% of the total phospholipid), is capable of
facilitating the transport of mucus by the movement of the cilia of the
respiratory mucosa or by the phenomenon of cough by altering the
visco-elastic properties of the mucus (reference: Galabert et al, Clin.
Chim. Acta, 1987, 164:139-149).
We have studied the role of the different phospholipid fractions, known to
reduce the surface tension at the alveolar level in the alterations of the
surface-active properties of the mucus at the air passages. The works
began with a study of patients affected with cysticfibrosis and chronic
obstructive bronchopathy, in whom the content of phospholipid fractions in
the expectorations and the surface properties of the phospholipid
fractions were measured in parallel. The relationship between the
biochemical data and the physical properties of the expectorations studied
was analysed.
The collection of the expectorations was carried out avoiding any salivary
contamination. The patients were divided into two groups composed of
eighteen with cysticfibrosis and of sixteen with chronic bronchitis with
hypersecretion and obstruction. The lipids were separated and identified
using conventional techniques; for this, the expectorations were
immediately frozen, then freeze-dried: the extraction of lipids was
carried out on freeze-dried material.
The quantitative extraction of the lipids was carried out according to the
method of Bligh E. G. and Dyer W. J.: A rapid method of total lipid
extraction and purification. Can. J. Biochem. Physiol., 1959, 37, 911-917:
100 mg of freeze-dried material are rehydrated in 1 ml of distilled water.
The lipids were then extracted with a chloroform/methanol mixture (1 v/2
v), then in a second chloroform/methanol mixture (1 v/1 v). After addition
of water, the chloroform phase was separated off. Three complementary
extractions by addition of 2.5 ml of chloroform were then carried out. The
different chloroform phases were then combined, evaporated to dryness
under nitrogen and P.sub.2 O.sub.5, and then weighed before resuspending
in solution in chloroform.
The total phospholipids and the phospholipid fractions, separated by
two-dimensional thin layer chromatography, were determined by the method
of Rouser et al. (Lipids, 1970, 5, 494-496) the principle of which is the
micro-determination of phosphorus after mineralisation of the
phospholipids by perchloric acid and separation by two-dimensional thin
layer chromatography.
From the determination of the phospholipids in these secretions, it was
shown that the total phospholipids were significantly higher in
cysticfibrosis than in chronic obstructive bronchitis (4.40% against 2.96%
of dry weight of mucus respectively (p<0.01)). The relative percentage of
phosphatidylcholine and phosphatidylglycerol was significantly lower
(p<0.001) in cysticfibrosis than in chronic obstructive bronchopathy
(Table I). The relative percentages of sphingomyelin and
phosphatidyl-serine+phosphatidylinositol were significantly higher
(p<0.001) in cysticfibrosis than in chronic obstructive bronchitis (Table
I).
TABLE I
______________________________________
Percentage of the main phospholipid fractions
relative to the total lipids
PG PC SM PS + PI
______________________________________
Cystiofibrosis 4.41 36.32 22.24
15.10
Chronic bronchitis
7.48 46.47 12.44
11.76
______________________________________
PC:phosphatidylcholine; PG:phosphatidylglycerol; SM:sphingomyelin;
PS+PI:phosphatidylserine+phosphatidylinositol.
In the case of cysticfibrosis, where the secretions are very adhesive and
very viscous, fewer surface-active fractions (phosphatidylcholine,
phosphatidylglycerol) and more "rigidifying" fractions (sphingomyelin)
were found in the expectorations than in the other bronchopathies.
A physical study of these secretions has been carried out in parallel. The
physical property chosen was the wettability of the secretions which
provides information on the ability of a secretion to wet a surface. The
wettability is estimated by a physical parameter called the contact angle.
The contact angle can be defined as the angle between the tangent to a
liquid droplet placed on a solid base (at the liquid-air interface) and
the surface of the solid (at the solid-liquid interface) at the triple
point Tp.
The contact angle depends on the solid, the nature of the liquid and the
interaction between them. We have studied the behaviour of bronchial
secretions when they are put into contact with a support which simulates
the bronchial mucosa. The contact angle is an index of wettability which
provides information on the surface-active properties of the liquid for a
given solid: the larger the contact angle, the less the liquid spreads and
the less wetting it is.
A technique for automatically measuring the contact angle based on image
analysis coupled with computer analysis has been developed. During
measurements of the wettability of the secretions, it was chosen to
measure the contact angle of a calibrated (5 .mu.l) secretion droplet
which was put into contact with a perfectly plain glass support,
electronegatively charged and placed in a container saturated with water
vapour in order to avoid dehydration processes (reference: S. Girod et al.
I.T.B.M, 1988, 9 402-412).
The phosphatidylglycerol appears to be an important phospholipid which
controls the wettability of bronchial secretions and which is capable of
influencing their transport and their behaviour when they are in contact
with the mucosa. We therefore studied the influence of various
phospholipids on the wettability properties of the respiratory mucus and
on its ability to be mobilised and transported by the mechanism of cough.
An experiment intended to study the effect, in vitro, of various
phospholipid fractions on the wetting ability and the transport by cough
of mucus considered as "normal" was therefore carried out.
The mucus was taken from frog palate mucosa which is considered to be a
good representative model of human respiratory mucosa. The wettability of
these mucus was measured as previously described. The clearance of these
mucus by cough was measured using a cough machine. The principle of this
machine is as follows: the support on which the mucus droplet is deposited
is used as the base for a plexiglas tube which simulates the trachea. The
plexiglas tube of rectangular section (20 mm.times.10 mm) is connected to
a container which is closed by means of an electrovalve. The pressure
inside the container is brought to 0.6 bar, then the electrovalve is
opened and the air flow rate to which the droplet is subjected is 6 l/s.
The displacement of the droplet on the support under the influence of the
air current thus produced, which simulates the mechanism of cough, is
measured.
The supports used were either simple glass plates or glass plates covered
with a monolayer of phospholipids using a Langmuir film balance: 10 mg of
each phospholipid fraction was dissolved in a chloroform/methanol mixture
(9:1) and 16 .mu.l of this solution was deposited in the basin of the film
balance filled with triple-distilled water. A Teflon barrier compressed
the phospholipid film until obtention of the collapsing pressure specific
to each phospholipid, at which all the phospholipid molecules are in
contact without holes in the monolayer. The glass support previously
immersed in the basin was then withdrawn at a constant speed and, at the
same time, the Teflon barrier advances so as to keep constant also the
surface tension of film thus removed; the glass support thus obtained had
on its surface an oriented monolayer of phospholipids attached to the
glass by their polar heads and exposing their non-polar groups to the air
(in contact with the mucus).
The phospholipids chosen for this study were phosphatidylglycerol
(diC.sub.18:1) (Sigma 9399) and phosphatidylglycerol (diC.sub.18:0) (Sigma
9524): these fractions therefore had the same polar head, the same fatty
acid chain length but differ from each other in the degree of saturation.
Glass plates not covered with phospholipids were kept as controls.
It emerges that phosphatidylglycerol (diC.sub.18:0) significantly improves
the clearance of frog mucus by cough and significantly decreases its
wettability, in comparison with uncovered glass plates (p<0.001 and
p<0.01) or even with glass plates covered with phosphatidylglycerol
(diC.sub.18:0) (p<0.001 and p<0.01). The phosphatidylglycerol
(diC.sub.18:0) appears to be the phospholipid fraction which, by reducing
the adhesive properties of the mucus, improves its clearance by cough (the
most lubricating fraction).
Phosphatidylcholine in the form of dipalmitoylphosphatidyl-choline
(diC.sub.16.0) was also studied with respect to the wettability and the
transporting ability by cough.
In view of these studies, the invention comprises a composition for use in
the treatment of the obstruction of air passages comprising one or more
phospholipids, alone or in association with excipients. The phospholipid
or phospholipids may be chosen from among phosphatidylcholines,
phosphatidylglycerols and phosphatidic acids.
The phospholipids use in accordance with the invention may comprise
identical or different, saturated or unsaturated acyl radicals.
The phospholipids used in the compositions in accordance with the invention
more particularly comprise two identical saturated acyl radicals.
The above mentioned phospholipids preferably comprise, by way of acyl
radicals, the saturated radicals palmitoyl C.sub.16:0 and stearoyl
C.sub.18:0 and/or the unsaturated radicals oleoyls C.sub.18:1 and
C.sub.18:2 ; and most preferably comprise dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, phosphatidyl-glycerol diC.sub.18:0,
phosphatidyl-glycerol diC.sub.18:1 and phosphatidylglycerol diC.sub.16:0.
The acyl radicals of the phospholipids may optionally contain several
double bonds.
The phospholipids may be used alone or, preferably, mixed with one another
in variable proportions.
The compositions in accordance with the invention may be provided in the
form of micellar suspensions or in the form of liposome suspensions. They
can be administered orally or aerially, for example by inhalation (in the
form of aerosols) or by instillation.
The compositions in accordance with the invention may also contain
therapeutic agents known for their activity in the field of
bronchopulmonary diseases and which are capable of having a specific
activity on the bronchial mucosa.
In particular, it is possible to combine the phospholipids, in the form of
micellar suspensions (or emulsions) or in the form of liposome
suspensions, as instillations or as aerosols, with the following
compounds:
agents protecting against free radicals such as cysteine derivatives or
superoxide dismutase,
antibacterial substances such as lysozyme or asialo-GMI-ganglioside
growth factors such as insulin, retinoic acid derivatives, epithelium
growth factor (or EGF) and platelet derived growth factor (or PDGF),
repair factors such as glycyl-L-histidyl-L-lysine and other peptides
purified from epithelial or mesenchymatous cells,
mucus-hydrating agents such as ATP.
The compositions of the invention are used for the treatment of
bronchopulmonary diseases.
The invention also relates to a method of treating the obstruction of
respiratory airways in a patient, which comprises administering to the
patient a therapeutically effective amount of a composition comprising one
or more phospholipids, as described above.
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